DESCRIPTION: (Applicant's Abstract) The cellular signaling mechanisms of the neuronal CB1 and peripheral CB2 cannabinoid receptor will be examined by evaluating the effectors that couple to these two receptors and the G-protein alpha subunits that mediate receptor-effector coupling. Adenylyl cyclase, Ca2+ channels and phospholipase C will be evaluated as the effectors. We will then test the hypothesis that G proteins of the Galpha-i and Galpha-o are mediators for the signalling of the cannabinoid receptor. The hypothesis will be tested by three complementary approaches. 1) The Galpha subunits that are activated upon stimulation of the cannabinoid receptor will be identified by photolabeling with the GTP analog, [alpha-32P]GTP azidoanilide, and subsequent immunoblotting with a panel of anti-Galpha antibodies. 2) The specific Galpha subunits that couple the receptor to the various effectors will be evaluated by using antisense RNA or antisense oligodeoxynucleotides directed against various Galpha subunits. 3) The specific Galpha subunits involved in coupling will then be confirmed with antibodies raised against different Galpha subunits which will interfere with the link in receptor -> G protein coupling. These three approaches will be used in two types of cellular models: NG108, GH4C1 and HL60 cells that express endogenous CB1 and CB2 cannabinoid receptors; and CHO and COS7 cells that express the receptors heterologously. The goal is to test whether there is any specificity in receptor -> G protein -> effector coupling, by comparing the signaling mechanisms of two cannabinoid receptors in different cellular environment.